The botulinum neurotoxins are potent neuroparalytic agents which pose a threat to human health both in natural outbreak occurrences and through deliberate release by bioterrorists. The currently accepted method of assay for the botulinum toxins, the mouse bioassay, is non-specific and too slow to provide a reliable diagnostic tool for use in an emergency response capacity. Rapid, sensitive and reliable in vitro assays are urgently required. Through a better understanding of the enzymology of the botulinum neurotoxins, the proposed study aims to develop sensitive in vitro assays for all seven toxin serotypes which can replace the mouse test. Target characteristics for the assay are:-- assay sensitivities equivalent to the mouse bioassay - applicable to a wide range of media including food extracts and serum- very low incidence of either false positive or false negative results - assay result in less than 6 hours. To achieve the required sensitivity (as low as 10 picograms/ml for botulinum neurotoxin serotypes A and B), assays will be developed which exploit a biological activity essential to the action of each neurotoxin. In these assay systems, the unique endopeptidase activity contained within the light chain of each botulinum neurotoxin will be used to amplify the assay to the desired sensitivity. This approach offers a number of advantages. Firstly, since the amplification system relies on the toxin itself, the requirement for specialized assay reagents is minimized. Secondly, since the assay signal depends upon a unique endopeptidase activity, the incidence of false-positive results will be very low. Lastly, since the assay is based on an essential biological activity within the neurotoxin, it closely resembles the mouse bioassay in that denatured toxin will not be detected. A prime goal of the research is to study further the enzymatic properties of the botulinum toxins and through this research develop endopeptidase-based, in vitro assays for each of the toxin serotypes and combine these into a simple, reliable test protocol.